CONSTRUCCION, ENSAYOS, DOCUMENTACION Y HABILITACION DE LAS INSTALACIONES
SUELOGas Natural
5.2.2 Unión de tuberías Uniones roscadas
Biochemical Composition of an Antarctic Chlorella
W.L. Chu1,2, S.M. Phang2*, S.L. Lim2, M.L. Teoh2 and C.Y. Wong2
Chlorella is one of the common microalgae found in a wide range of habitats, including Antarctica. Chlorella UMACC 234 is an interesting isolate in the collection of Antarctic microalgae in the University of Malaya algae culture collection (UMACC) as it grows well at temperatures much higher than the ambience. The alga was isolated from snow samples collected from Casey, Antarctica. This study investigates the influence
of nitrogen source on the growth, biochemical composition and fatty acid profile of Chlorella UMACC
234. The cultures were grown in Bold’s Basal Medium with 3.0 mM NaNO3, NH4Cl or urea. The cultures
grown on NaNO3 attained the highest specific growth rate (µ = 0.43 day–1) while the specific growth rates of
those grown on NH4Cl and urea were not significantly different (p > 0.05). The urea-grown cells produced
the highest amounts of lipids (25.7% dry weight) and proteins (52.5% dry weight) compared to those grown
on other nitrogen sources. The cell numbers attained by the cultures grown at NaNO3 levels between 0.3 and
3.0 mM were similar but decreased markedly at 9.0 mM NaNO3. The fatty acids of Chlorella UMACC 234
were dominated by saturated fatty acids, especially 16:0 and 18:0. The percentage of polyunsaturated fatty acids was very low, especially in cells grown on urea (0.9% total fatty acids). Characterisation of the growth and biochemical composition of this Antarctic Chlorella is important to our studies on the relationship of Chorella isolates from tropical, temperate and polar regions, especially in terms of phylogeny and stress adaptation.
Key words:Chlorella; Antarctica; nitrogen source; biochemical composition; fatty acids; urea
1International Medical University, No. 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia
2Institute of Biological Sciences
& Institute of Earth and Ocean Science, University of Malaya, 50603 Kuala Lumpur, Malaysia *Corresponding author (e-mail: [email protected])
W.L. Chu et al.: Influence of Nitrogen Source on the Growth and Biochemical Composition of an Antarctic Chlorella
179 soils highly enriched with nitrogen and phosphorus while chlorophytes and diatoms were dominant in sites with lower amounts of these two nutrients in a study conducted at Cierve Point, Antarctic Peninsula (Mataloni & Tell 2002).
The objective of the present study was to investigate the influence of nitrogen source on the growth, biochemical
composition and fatty acid profile of Chlorella UMACC
234. The study was important as enrichment of nitrogen could affect the adaptation and occurrence of algae in the harsh environment of Antarctica.
MATERIALS AND METHODS
Algal Cultures
The cultures of Chlorella UMACC 234 were maintained in Bold’s Basal Medium (BBM) (Nichols 1973) and grown in a controlled-environment incubator set at 4ºC with irradiation
of 42 µmol m–2 s–1 on a 12:12 h light-dark cycle.
Effect of Nitrogen Source
The inoculum from exponential phase cultures standardised
at an optical density at 620 nm (OD620) of 0.2 were used
for all the experiments. Three nitrogen sources were
used, namely NaNO3, NH4Cl and urea. A volume of 10
mL of culture was inoculated into 90 ml BBM containing
3 mM of NaNO3, NH4Cl or urea, buffered with 10 mM
4-(2-hydroxyethyl)-piperazine-1-ethane-sulphonic acid (HEPES). The concentration of nitrogen source used was
equivalent to the NaNO3 contained in BBM. The cells from
the inoculum were pelleted by centrifugation (3000 r.p.m., 20 min) and resuspended in nitrogen-free medium before being added into BBM with the different nitrogen sources. After inoculation, growth was monitored by counting the cells daily for 10 days using a haemocytometer (Improved Neubauer).
The specific growth rate (µ, day–1
) was determined
using the following formula: µ = (ln N1 – ln N2) / (t2 – t1)
where N1 and N2 represent the cell number at times t1 and t2
respectively, within the exponential phase.
At the end of the experiment, the cells were harvested by filtration onto glass fibre filters (4.7 cm, 0.45 µm) for the extraction of lipids, carbohydrates, proteins and chlorophyll
a. Lipids were extracted in MeOH-CHCl3-H2O (2:1:0.8)
and determined by gravimetric method (Bligh & Dyer 1959). Proteins were extracted in 0.5 N NaOH (80ºC, 30 min) and the concentration determined by the dye-binding method (Bradford 1976). Carbohydrates were extracted in 2 N HCl (80ºC, 30 min) and the concentration determined by the phenol-sulphuric method (Kochert 1978). The lipids
were transesterified in 1% H2SO4 in methanol and the fatty
acid methyl esters were analysed by gas chromatography as described in Chu et al. (1994).
Effect of NaNO3 Level
In this experiment, the cultures were grown in BBM
containing 0, 0.3, 1.0, 3.0 and 9.0 mM NaNO3 buffered
with 10 mM HEPES. Growth was monitored based on cell number on day 4 and 8.
Statistical Analysis
The data were compared using one-way ANOVA followed by Duncan’s multiple range test (Statistica v5.0). The difference between means was considered significant when p < 0.05.
RESULTS
Of the three nitrogen sources, NaNO3 supported the best
growth of Chlorella UMACC 234 based on specific growth rate (µ) and final cell number, biomass and chlorophyll a concentration attained (Table 1). The µ’s of the cultures
grown on NH4Cl and urea did not differ significantly (p >
0.05). Further experiments were conducted to investigate
the effect of NaNO3 levels on the growth of Chlorella
UMACC 234. The growth of Chlorella UMACC 234 was
similar at NaNO3 concentrations ranging from 0.3 mM to
3.0 mM (Figure 1). The cell number decreased markedly at
9.0 mM NaNO3.
In terms of biochemical composition, cells grown on urea contained the highest amount (p < 0.05) of proteins (52.5% dry weight) (Figure 2). In comparison, cells grown
on NaNO3 contained the highest amount (p < 0.05) of
carbohydrates (30.4% dry weight). The lipid content ranged from 12.3% to 25.6% dry weight and was highest in cells grown on urea.
The dominant group of fatty acids of Chlorella UMACC 234 was saturated fatty acids (SFA), ranging from 63.7% to 96.7% total fatty acids (Figure 3). The major SFA of Chlorella UMACC 234 were 16:0 and 18:0 (Table 2). The percentage of polyunsaturated fatty acids (PUFA) with the
dominance of 18:3 was highest in cells grown on NH4Cl.
Cells grown on urea contained a very low percentage of PUFA.
DISCUSSION
The ability of Chlorella to utilise different nitrogen sources varies with species and strains. For instance, endosymbiotic Chlorella strains in Parameciumbursaria were not able to utilise ammonium nitrate as the sole nitrogen source, but were able to assimilate a wide range of amino acids (Kato
180
Table 1. Growth characteristics of Chlorella UMACC 234 cultured on different nitrogen sources.
Specific growth rate Final cell number Final biomass Final chlorophyll Nitrogen source (µ, day–1
) (x 108 mL–1) (mg dry weight L–1) a concentration
(mg L–1)
NaNO3 0.43 ± 0.02a* 7.36 ± 0.30a 185.0 ± 10.0a 1.22 ± 0.06a
NH4Cl 0.36 ± 0.01b 5.22 ± 0.15b 161.0 ± 3.0a 0.88 ± 0.05b
Urea 0.31 ± 0.05b 5.05 ± 0.64b 103.0 ± 22.0b 0.70 ± 0.10b
*Different alphabets within the same column denote significant differences at p < 0.05 (n=3)
Table 2. Fatty acid composition (% total fatty acids) of Chlorella UMACC 234 grown on different nitrogen sources. All values are
expressed as mean ± standard deviation.
Fatty acid Nitrogen sourceNaNO
3 NH4Cl Urea
Saturated fatty acids (SFA)
14:0 4.3 ± 0.5 – 2.7 ± 1.3
16:0 54.8 ± 6.1 45.8 ± 0.1 49.9 ± 5.2
18:0 24.0 ± 0.1 22.1 ± 5.4 44.1 ± 2.5
Monounsaturated fatty acids (MUFA)
16:1 0.5 ± 0.1 – –
18:1 3.5 ± 1.1 7.5 ± 0.6 2.9 ± 0.6
Polyunsaturated fatty acids (PUFA)
16:4 2.6 ± 0.9 2.6 ± 0.6 – 18:2 0.7 ± 0.1 4.1 ± 5.9 – 18:3 9.2 ± 1.6 17.3 ± 5.9 0.9 ± 0.1 18:4 1.5 ± 0.1 1.4 ± 0.1 – 0 50 100 150 200 250 0 0.3 1.0 3.0 9.0 NaNO3 concentration (mM) x 10 6 cells mL −1 Day 4 Day 8 a a b b c b b b a a
Figure 1. Growth based on cell number of Chlorella UMACC 234 cultured at different concentrations of NaNO3. Different alphabets
above the bar charts denote significant differences (p < 0.05, n = 3) in the cell numbers on day 4 and day 8, respectively.
0 10 20 30 40 50 60 NaNO3 NH4Cl Urea Nitrogen source % D ry w e ig h t Proteins Carbohydrates Lipids a a a a b a b b b 0% 20% 40% 60% 80% 100% NaNO3 NH4Cl Urea Nitrogen source
% Total fatty acids
PUFA MUFA SFA
Figure 2. Biochemical composition of Chlorella UMACC 234 grown on different nitrogen sources. Vertical bars indicate standard deviations from three replicates. Different alphabets above the bar charts for each
biochemical component denote significant differences at p < 0.05 (n = 3).
Figure 3. Distribution of saturated, monounsaturated and polyunsaturated fatty acids (SFA, MUFA and PUFA)
182
et al. 2006). In comparison, Chlorella prothecoides grew
better on NH4+ than on NO3- (Ahmad & Hellebust 1990).
Some Chlorella strains might be highly tolerant to ammoniacal nitrogen. For instance, an isolate of Chlorella pyrenoidosa from leachate samples was able to grow, even
at NH4+-N as high as 135 mg L–1 (Lin et al. 2007). Results
showed that Chlorella UMACC 234 grew better on
NaNO3 than NH4Cl or urea. This is in contrast with other
Chlorella strains which grow well on urea. For instance, C. protothecoides produced higher biomass when grown on
urea than on NO3- or NH4+ under heterotrophic condition
(Shi et al. 2000).
The lower growth on ammonium and urea compared to nitrate could be due to the origin of this alga. Chlorella UMACC 234 was isolated from snow, which is known to contain very little ammonium or urea. Snow algae are known to be psychrophilic and they do not grow at temperatures above 10ºC (Hoham 1975). However, Chlorella UMACC 234 grows even at 30ºC (Teoh et al. 2004). Thus, it is most probably a soil rather than a snow alga. Soil algae are known to be brought onto the snow surface due to wind action, as reported for Raphidonema nivale (Stibal & Elster 2005). The soil below the snow at the collection site of Chlorella UMACC 234 was probably low in nitrogen. This was different from other soil habitats such as penguin rookeries which may contain high levels of ammonium and nitrate (Mataloni & Tell
2002; Ohtani et al. 2000). However, in those habitats,
chlorophytes such as Stichococcus bacillaris and Prasiola crispa are commonly found but not Chlorella. In contrast, several species of Chlorella have been found to occur in mineral soils low in nitrogen at Victoria Land, Antarctica (Cavacini 2001) and Cierva Point, Antarctic Peninsula (Mataloni et al. 2000).
Chlorella UMACC 234 grew well at NaNO3 concentrations ranging from 0.3 mM to 3.0 mM but its growth was markedly reduced at 9.0 mM. The Antarctic Chlorella isolate required a much lower concentration
of NaNO3 for growth compared to the tropical isolate
Chlorella vulgaris UMACC 001, which grew well even at
18.75 mM NaNO3 (Chu et al. 2007).
The major biochemical component of Chlorella UMACC 234 were proteins (33.5% – 52.5% dry weight) followed by carbohydrates (16.8% – 30.4% dry weight)
and lipids (12.3% – 25.6% dry weight). Chlorella is known
to contain high amounts of protein. For instance, Chlorella sorokiniana, an isolate from hot springs, contains 68.5% protein (Matsukawa et al. 2000).
The nitrogen source is an important factor that can influence the biochemical composition of algae; however, the effect varies with species. For instance, the marine eustigmatophyte Ellipsoidion sp. contains higher amounts
of lipids when grown on NH4Cl, than on NaNO3 or urea
(Xu et al. 2001). Chlorella UMACC 234 grown on urea produced higher amounts of lipids and carbohydrates at the
expense of proteins compared to cells grown on NaNO3
and NH4Cl. However, the protein content of cells grown
on NaNO3 and NH4Cl were not significantly different. This
was in contrast with C. protothecoides, which contains
more proteins in cells grown on NO3- than those on NH4+
(Ahmad & Hellebust 1990).
The fatty acid composition of Chlorella UMACC 234 was similar to that of other Chlorella strains, with the
dominance of SFA, especially 16:0 and 18:0 (Teoh et al.
2004; Wong et al. 2007). In comparison, 18:0 was not found
in three species of temperate Chlorella and a psychrophilic strain of Chlorella as reported by Petkov and Garcia (2007) and Morgan-Kiss et al. (2008), respectively. The high
percentage of 18:0 detected in Chlorella UMACC 234
could be due to the use of non-aerated cultures. Aeration
is known to affect the production of 18:0 in Chlorella. For
instance, a high percentage of 18:0 was only produced in C. sorokiniana when the cultures are not aerated (Chen & John 1991). Urea also appeared to enhance the production
of 18:0 at the expense of 18:3 in Chlorella UMACC 234.
A high percentage of 18:0 was reported for C. pyrenoidosa
grown in olive-mill wastewater rich in organic nitrogen
source (Sanchez et al. 2001). There was a relatively low
percentage of PUFA in this Chlorella, in agreement with
the findings on other Antarctic Chlorella strains (Wong
et al. 2007; Morgan-Kiss et al. 2008).
The characterisation of Chlorella UMACC 234 in terms of nitrogen requirement would contribute to our studies on the response of Antarctic Chlorella to stress, in comparison with temperate and tropical isolates. The findings would also contribute to our studies on phylogeography of Chlorella from different regions.
ACKNOWLEDGEMENTS
This study was supported by a research grant from the Ministry of Science, Technology and Innovation, Malaysia co-ordinated by the Academy of Sciences Malaysia. This research also forms part of a project under the Australian Antarctic Division (AAD #2694). The berths offered by the AAD and field assistance provided by the staff of Casey Station are gratefully acknowledged.
Date of submission: October 2008 Date of acceptance: November 2009
REFERENCES
Ahmad, I & Hellebust, JA 1990, ‘Regulation of chloroplast development by nitrogen source and growth conditions in a Chlorella prothecoides strain’, Plant Physiology, vol. 29, 294 – 300.
W.L. Chu et al.: Influence of Nitrogen Source on the Growth and Biochemical Composition of an Antarctic Chlorella
183
Bligh, EG & Dyer, WJ 1959, ‘A rapid method of total lipid extraction and purification’, Canadian Journal of Biochemistry
and Physiology, vol. 37, pp. 911–917.
Bradford, MM 1976, ‘A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of dye-binding’, Analytical Biochemistry, vol. 72, pp. 248–254.
Cavacini, P 2001, ‘Soil algae from northern Victoria Land (Antarctica)’, Polar Bioscience, vol. 14, 45–60.
Chen, F & John, MR 1991, ‘Effect of C/N ratio and aeration on the fatty acid composition of heterotrophic Chlorella sorokiniana’, Journal of Applied Phycology, vol. 3, pp. 203– 209.
Chu, WL, Phang, SM & Goh, SH 1994, ‘Studies on the production of useful chemicals, especially fatty acids in the marine diatom Nitzschia conspicua Grunow’, Hydrobiologia, vol. 285, pp. 33–40.
Chu, WL, Ramadhona, MS & Phang, SM 2007, ‘Assessment of three tropical chlorophytes as bioassay organisms for nitrogen and phosphorus enrichment in freshwater ecosystems’, Malaysian Journal of Science, vol. 26, no. 2, 15–25.
Chu, WL et al. 2002, ‘Isolation and culture of microalgae from the Windmill Islands Region, Antarctica’, in Proceedings of the Malaysian International Seminar on Antarctica, 5–6 Aug 2002, Kuala Lumpur, pp. 53–59.
Hoham, RM 1975, ‘Optimum temperatures and temperature ranges for growth of snow algae’, Arctic Alpine Research, vol. 7, pp. 13–24.
Kato, Y, Ueno, S & Imamura, N 2006, ‘Studies on the nitrogen utilization of endosymbiotic algae isolated from Japanese Paramecium bursaria’, Plant Science, vol. 170, no. 3, pp. 481–486.
Kochert, AG 1978, ‘Carbohydrate determination by the phenol-sulfuric acid method’, in Handbook of phycological
methods: physiological and biochemical methods, eds
JA Hellebust & JS Craig, Cambridge University Press, pp. 95–97.
Lin, L et al. 2007, ‘Use of ammoniacal nitrogen tolerant microalgae in landfill leachate treatment’, Waste
Management, vol. 27, pp. 1376–1382.
Mataloni, G & Tell, G 2002, ‘Microalgal communities from ornithogenic soils at Cierva Point, Antarctic Peninsula’, Polar Biology, vol. 25, pp. 488–491.
Mataloni, G, Tell, G & Wynn-Williams, DD 2000, ‘Structure and diversity of soil algal communities from Cierva Point (Antarctic Peninsula)’, Polar Biology, vol. 23, pp. 205–211.
Matsukawa, R et al. 2000, ‘Antioxidants from carbon dioxide fixing Chlorella sorokiniana’, Journal of Applied Phycology, vol. 12, pp. 263–267.
Morgan-Kiss, RM et al. 2008, ‘Identity and physiology of a new psychrophilic eukaryotic green alga, Chlorella sp., strain BI, isolated from a transitory pond near Bratina Island, Antarctica’, Extremophiles, vol. 12, pp. 70 –711.
Nichols, HW 1973, ‘Growth media – freshwater’, in Handbook of phycological methods: culture methods and growth measurements, ed J Stein, Cambridge University Press, pp. 7–24.
Ohtani, S et al. 2000, ‘Distribution of soil algae at the monitoring sites in the vicinity of Syowa Station between austral summers of 1992/1993 and 1997/1998’, Polar Bioscience, vol. 13, pp. 113–132.
Petkov, G & Garcia, G 2007, ‘Which are fatty acids of the green alga Chlorella?’ Biochemical Systematics and Ecology, vol. 35, pp. 281–285.
Phang, SM & Chu, WL 2004, ‘The University of Malaya Algae Culture Collection (UMAC) and potential applications of a unique Chlorella from the collection’, Japanese Journal of
Phycology, vol. 52, pp. 221–224.
Phang, SM et al. 2007, ‘A checklist of microalgal isolates from Ny Alesund, Svalbard’, in 8th Ny-Alesund Seminar, 16 – 17 October 2007, Cambridge, UK, The Journal of the CNR’s
Network of Polar Research, pp. 11–14.
Sanchez, S et al. 2001, ‘Mixotrophic culture of Chlorella
pyrenoidosa with olive-mill wastewater as the nutrient
medium’, Journal of Applied Phycology, vol. 13, pp. 443– 449.
Shi, X, Zhang, X & Chen, F 2000, ‘Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources’, Enzyme and Microbial Technology, vol. 27, pp. 312–318.
Stibal, M & Elster, J 2005, ‘Growth and morphology variation as a response to changing environmental factors in two Arctic species of Raphidonema (Trebouxiophyceae) from snow and soil’, Polar Biology, vol. 28, pp. 558–567.
Strickland, JD & Parsons, TR 1968, A practical handbook of
seawater analysis, Bulletin No 167, Fisheries Research Board
Canada.
Teoh, ML et al. 2004, ‘Influence of culture temperature on the growth, biochemical composition and fatty acid profiles of six Antarctic microalgae’, Journal of Applied Phycology, vol. 16, pp. 421–430.
Wong, CY et al. 2007, ‘Comparing the response of Antarctic, tropical and temperate microalgae to ultraviolet radiation (UVR) stress’, Journal of Applied Phycology, vol. 19, pp. 689– 699.
Xu, N et al. 2001, ‘Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta)’, Journal of Applied Phycology, vol. 13, pp. 463–469.
184 Antarctic Microfungal Diversity
Studies of the diversity of microfungi in the Antarctic has predominantly focused on the Continental Antarctic (Sugiyama et al. 1967; Wicklow 1968; Atlas et al. 1978; Friedmann 1982; Martin 1988; Del Frate & Carretta 1990; Onofri & Tosi 1992; Moller & Gams 1993; Smith 1994; Azmi & Seppelt 1998; Cheryl & Seppelt 1999; Selbmann et al. 2005) rather than maritime Antarctic (Dennis 1968; Gray et al. 1982; Pugh & Allsopp 1982; Weinstein et al. 1997). Studies of fungi in the barren soils of the Antarctic have included areas that are more easily accessible such as the Windmill Islands (Azmi & Seppelt 1990; Cheryl & Seppelt 1999), and more challenging areas such as the Victoria Land Dry Valleys (Friedmann et al. 1985; Cameron et al. 1971). The occurrence of fungi in areas of
historic human activity — the ‘historic huts’ and associated habitats — have been studied extensively by Tubaki (1961), Martin (1988), Blanchette (2000), Blanchette et al. (2004) and Held et al. (2005).
Fungal studies have encompassed a range of habitats, including soil (Onofri 1990; Kerry 1990a,b; Finotti 1992; Azmi & Seppelt 1998; Hughes et al. 2003), ice and perma- frost (Gilichinsky et al. 2005), lake sediments (Sugiyama et al. 1967) and also the air (Marshall 1997). Airborne transfer of spores of Cladosporium sp. between South America and the Maritime Antarctica have been proposed (Marshall 1997). A number of fungi reported in Antarctic studies do not appear to have growth characteristics in culture that suit them well to the Antarctic environment. For instance, Sugiyama et al. (1967) reported Penicillium